The long range objective of the proposed research is to discover new mechanisms of regulation of enzyme activity, mechanisms that operate in addition to modification of enzyme activity by non-covalent binding of effector molecules and repression of enzyme synthesis. In particular, we are seeking control mechanism that may be related to enzyme turnover and inactivation, processes that may be important during development and differentiation. The specific experimental system to be analyzed is the biochemical fate of the enzymes of de novo pyrimidine and purine biosynthesis during growth and sporulation of Bacillus subtilis. These synthetic capacities are lost during endospore formation. In the case of de novo pyrimidine synthesis, aspartic transcarbamylase has been shown to be rapidly inactivated at the end of log phase growth, in a process that requires metabolic energy, but which is not blocked by antibiotics that inhibit protein synthesis. This proposal contemplates learning the biochemical fate of aspartic transcarbamylase during sporulation by a combination of approaches including use of specific metabolic inhibitors, mutant strains, in vitro inactivation studies, and purification of the enzyme from vegetative and sporulating cells. The regulation of inactivation will be studied by physiological and biochemical techniques. In the case of de novo purine synthesis, the first enzyme of the pathway, PRPP:glutamine amidotransferase has also been shown to be rapidly inactivated at the end of exponential growth in a process that apparently involves molecular oxygen rather than metabolic energy.